Biol 568 Advanced Topics in Molecular Genetics

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Biol 568Advanced Topics in Molecular Genetics
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Ch. 23 Controlling Chromatin Structure
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• Chromatin remodeling and activation
• Remodeling complexes
• Txn factors
• Histone Modifications
• DNA methylation
• Active and inactive chromatin

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Control of gene expression

• Activation of chromatin structure
• Initiation of txn
• Txn processing
• Transport to cytoplasm
• Translation

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Gene expression analyses

• Three test systems
• Oocyte system
• Transfection systems
• Transgenic systems

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Chromatin can have alternative states

• Local chromatin
• Important part for gene expression
• The gene is in an active state only in the
  cells in which is expressed
• Epigenetic patterns of gene expression
• Heterochromatin formation

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Activation of Txn

• In prokaryotes
• Requires activator/ repressor
• Increase of activator/repressor concentration
  affects txn
• Equilibrium model

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Equilibrium model

• Fig. 23.1

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Activation of Txn

• In eukaryotes
• Requires specific activators/repressors
• Changes in the concentration of txn factor or
  repressor does not affect the chromatin structure
• Chromatin remodeling

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Chromatin remodeling

• Expression of 5S requires TFIIIA, RNA pol III
• In vitro
• Naked DNA -gt TFIIIA Txn
• Naked DNA -gt Histones -gtTFIIIA No Txn
• Naked DNA -gt TFIIIA -gt Histones Txn

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Chromatin remodeling

• Presence of TFIIIA prevents binding of
  nucleosomes to specific DNA regions

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Chromatin remodeling

• TFII D, RNA pol II
• Adenovirus promoter
• Histones added first No TXN
• TFIID added first TXN
• RNA pol II can be added later
• Nucleosomes form

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Chromatin remodeling

• TFII D creates a structure to which other txn
  components can bind

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Txn and chromatin remodeling

• Txn factors cannot recognize or cannot function
  on nucleosomal DNA
• The txn factors, once bound, remain to the site
• cannot be changed by disturbing the equilibrium

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Chromatin remodeling

• Fig. 23.2

• Txn factors or nucleosomes may form stable
  structures
• Cannot be altered by changing the equilibrium
  with free activator!

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Chromatin Remodeling

• The process of introducing changes in chromatin
  structure
• Disruption of DNA-protein, protein-protein
  contacts to release the histones from the
  chromatin

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Chromatin Remodeling

• Fig. 23.3

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Remodeling changes in chromatin

• Fig 23.4

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• Chromatin remodeling and activation
• Remodeling complexes
• Txn factors
• Histone Modifications
• DNA methylation
• Active and inactive chromatin

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Remodeling complexes
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SWI/SNF

• Yeast
• Interacts with chromatin to activate a subset of
  yeast genes
• 10 proteins
• Needed for expression of genes involved in
  mating-type switching and sucrose metabolism
  (sucrose non-fermenting)
• Contains ATPase activity
• Mammalian homologs hSWI/SNF
• ATPase is BRG1, related to Drosophila Brahma

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SWI/SNF

• Mutations are pleyotropic
• Similar to CTD deletions
• Suppressors of swi or snf mutants are mutations
  in genes encoding histones
• Acts catalytically in vitro
• In presence of ATP alters/displaces histone
  octamers
• Generates DNAse I sensitivity sites
• The remodeling energy is provided by SWI2
  susbunit
• Nonessential

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SWI/SNF txn

• The SWI/SNF complex is required for
  transcriptional activation of 5 yeast genes
• Can be recruited directly through interaction
  with DNA binding transcription factors
• Can be recruited indirectly by interaction with
  other txn coactivators or with the RNA polymerase
  holoenzyme

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Remodeling Txn factors

• How are remodeling complexes targeted to specific
  sites?
• TX factors

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Transcription factors

• Swi5
• Activates HO gene
• Binds to HO promoter
• Recruits SWI/SNF complex
• Swi5 disassociates from the regulatory sequence,
  SWI/SNF complex remains
• Activates txn by a hit and run mechanism!
• The role of TF is completed once the remodeling
  complex has bound

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Transcripton Factors

• GAGA factor
• Activates hsp70 promoter in vitro
• Binds to the promoter
• Introduces DNAse sensitive site
• Adjacent nucleosomes occupy preferential
  positions
• Positioning of the nucleosome at GAGA site
  altered

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PHO system

• PHO 4
• bHLH structure
• Responds to phosphate starvation
• Binds to PHO 5 promoter
• Repositions 4 nucleosomes
• Independent of txn and replication
• Two binding sites
• between nucleosomes and nucleosomal
• Activating region required for binding to
  nucleosomal site

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Remodeling complex is binds to site via activator

• Fig. 23.6

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TFs may bind to nucleosomal DNA

• TFs bind to DNA on outside of octamer
• Steroid receptor binding
• Nucleosomes positioned at precise sites
• Receptor binding alter DNA-histone interactions
  without displacement of nucleosomes
• Other factors then bind

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TFs bind to nucleosomal DNA

• MMTV promoter
• HRE Six palindromic domains
• Other NF1, OTF
• HR and NF1 cannot bind simultaneously to naked
  DNA
• Nucleosomal structure controls binding of all
  factors
• Rotational positioning required

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Fig 23.7 Requirement for nucleosomal organization
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• Chromatin remodeling and activation
• Remodeling complexes
• Txn factors
• Histone Modifications
• DNA methylation
• Active and inactive chromatin

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Histone modification

• Acetylation
• Deacetylation
• Methylation

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Histone modifications

• Fig 20.24

• Fig 20.26

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Histone modifications

• Fig. 23.8

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Effects of histone modifications
Fig 23.9-10

• Most histones have single modification
• Individual functions can be associated

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Histone Acetylation

• Major targets
• H3, H4 N-terminal domains
• DNA replication
• Transient acetylation of gistones during the S
  phase
• Acetylated transiently during tetramer formation
• Inhibition of acetylation in yeast is lethal

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Histone Acetylation

• Fig. 23.11

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Histone acetylation and txn activation

• Acetylation is increased in chromatin domains
  that contain active genes
• Sensitivity to DNAse I
• Acetylated nucleosomes are found at promoter
  vicinity of active genes
• Sex chromosome

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Histone acetylation and txn activation

• Fig. 23.12
• Acetyl groups
• Prerequisite for less condensed/ active chromatin
  structure

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Histone acetylation is reversible

• Acetylation
• HAT Histone acetyl transferases
• Group A txn
• Group B nucleosome assembly
• Deacetylation
• HDAC Histone deacetylases

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HAT Activity

• p300/CBP
• Higher eukaryotes
• Interacts with AP1, MyoD, c-Jun, c-Fos
• HAT activity, H4
• Inactivated by E1A and SV40T antigen

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HAT Activity
Fig 23.13
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HAT Activity

• GCN5
• Yeast regulator
• Important for enhancer-promoter interaction
• HAT activity, H3, H4
• Required for target activation

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HAT activity, part of holocomplex

• TFIID subunit
• TAF145 HAT activity
• GCN5 and TAF145 are lethal!
• Other HATs
• pCAF ( complex with p300/CBP)
• ACTR ( complex with hormone receptors)

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Histone Acetylation

• Gene activation
• Acetylation is maintained during gene activity
• A co-activating complex may contain multiple HAT
  activities
• Multiple acetylation events may be required

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HAT activity, overview
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Role of histone acetylation

• Qualitative and quantitative
• Transient in DNA replication
• Maintained in gene activation
• Necessary to loosen chromatine structure
• May facilitate incorporation of histones in
  nucleosomes ( replication)
• May facilitate structural changes or nucleosome
  displacment ( transcription)

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Histone deacetyation ( HDAC)
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Histone deacetyation

• Repression of txn
• Yeast
• DNA binidng protein Sin3 brings to regulatory
  regions Rpd3 ( HDAC)
• Mammalian
• Retinoid hormone repressors
• SMRT, mSIN3
• Heterochromatin
• Both constitutive and facultative

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Histone methylation

• H3 9Lys, associated with condensed chromatin
• Other positions, arginine,lysine
• May be connected with DNA methylation

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• Chromatin remodeling and activation
• Remodeling complexes
• Txn factors
• Histone Modifications
• DNA methylation
• Active and inactive chromatin

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DNA Methylation

• Methylation at promoter vicinity
• Absence of txn
• Applies to both alleles
• Occurs as an epigeneitc event
• Methylation assays
• Restriction enzymes
• Cleavage of sites containing CG doublets

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Methylation assays
Figure 21.25 MspI, Hpa II isoschizomers same
targets different responses
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Methylation assays

• Fig. 21.26

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DNA Methylation

• Demethylation can result in gene expression
• 5-azacytidine
• Changes the state of cellular differentiation
• Muscle cells from non muscle precursors
• Methylation connected with chromosomal activity

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DNA Methylation

• DNA introduced to host cells
• methylated gene inactive
• non methylated gene active
• a-globin gene cluster in chicken adult
  erythroids
• Undermethylated
• Undermethylated regions surround active genes
• Coincides with DNAse I sensitivity

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DNA Methylation

• Transcriptionally active genes are not methylated
  at 5
• Methylation is critical at specific sites
• g-globin ( -200, 90)
• Only a minority of methylation sites are involved
  in transcription

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DNA Methylation

• CpG - rich islands
• Only at 20 of expected
• Increased 10X in certain regions
• 45,000 CpG islands in human genome
• 1-2 kb streches
• CpG - rich islands
• several promoters, housekeeping genes

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Density of CpG doublets
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DNA Methylation

• Presence of CpG
• Gene potentially active
• Methylation of CpG prevents transcription
• MeCp1 MeCp2 bind to methylated CpG
• MeCp2 binds to Sin3 repressor complex

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DNA de-methylation

• Changes that occur in active genes
• Hs site(s) established near the promoter
• The nucleosomes of a domain including the
  transcribed region become sensitive to DNAse I
• The DNA of the same region is undermethylated

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DNA Histone methylation

• Methylation, histone deacetylation and
  methylation may be connected
• One event may trigger another

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Histones exist in alternative states
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Chromatin states are interconverted

• Fig. 23.16

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Remodeling Activation

• Fig. 23.17

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• Chromatin remodeling and activation
• Remodeling complexes
• Txn factors
• Histone Modifications
• DNA methylation
• Active and inactive chromatin

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Position effect variegation (PEV)

• Fig. 23.19
• The white gene is inactivated by neighboring
  heterochromatin in some cells

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Inactivate structure propagates

• Fig. 23.20
• Two step event
• Nucleation
• Propagation

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Heterochromatin

• Fig. 23.22

• DNA methylation
• Histones deacetylation and methylation
• Other proteins associated

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Heterochromatin protein 1 ( HP1)
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HP1 binding triggers silencing

• Fig. 23.24

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(No Transcript)
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Position effect variegation (PEV)

• Alpha and beta globin genes
• Erythroid cells
• T lymphocytes

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Chromatin analysis

• Treatment with histone deacetylase inhibitors,
• Butyric acid
• Trichostatin A

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Chromatin Analyses

• Preparation of mononucleosomes by micrococcal
  nuclease digestion
• Immunoprecipitation of mononucleosomes
• Separation of acetylated nucleosome
• DNA extraction
• Slot Blot, PCR, sequence analyses

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Chromatin Analyses
Micrococcal nuclease digestion
Mononucleosome isolation (centrifugation gradient)
Immunoprecipitation
Analysis of regulatory regions
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• Chromatin remodeling and activation
• Remodeling complexes
• Txn factors
• Histone Modifications
• DNA methylation
• Active and inactive chromatin